Treatment of oncologic tumors with an injectable formulation of a golgi apparatus disturbing agent

ABSTRACT

Novel pharmaceutical formulations for treating a cellular proliferative disease are provided comprising: a therapeutically effective amount of a Golgi apparatus disturbing agent; a biocompatible carrier; and a solvent. In preferred formulations, the Golgi apparatus disturbing agent is brefeldin A (BFA) and the biocompatible carrier is a polymer such as chitin or chitosan. Methods of treating cellular proliferative diseases using the pharmaceutical formulations are also described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.09/397,390, filed Sep. 15, 1999 now U.S. Pat. No. 6,287,602, whichclaims priority under 35 U.S.C. §119(e)(1) to U.S. ProvisionalApplication Serial No. 60/100,479, filed Sep. 16, 1998.

TECHNICAL FIELD

This invention relates to pharmaceutical formulations comprisingpharmacologically active agents, biocompatible carriers, and solventsand the like. More particularly, the invention relates to pharmaceuticalformulations containing Golgi apparatus disturbing agents such as, forexample, brefeldin A. This invention also relates to methods of treatingcellular proliferative diseases in patients in need of such therapy.

BACKGROUND

Local cancer chemotherapy involves the introduction of an anti-canceragent near or within a tumor. As a potential cure for some cancers,local chemotherapy has generated a tremendous interest among researchersand health care providers, in part because local chemotherapy (incontrast to systemic delivery) avoids or minimizes the potential forsystemic toxicity, and in part because the target site can be exposed tohigher concentrations of the active agent than possible withconventional chemotherapy. Thus, local chemotherapy can provide a usefultool in the treatment of some cancers.

Local chemotherapy is not, however, without drawbacks. One problemassociated with local chemotherapy is insufficient retention of thechemotherapeutic agent at the target site (i.e., the diseased organ ortissue). Another problem with local chemotherapy (and many types ofchemotherapy, for that matter) is the insoluble or slightly solublenature of the active agent. Thus, the ability of local chemotherapy tooffer a cure for some cancers has been compromised by retention and/orsolubility problems associated with previously known or suggestedchemotherapeutic agents and formulations.

The proposed solutions to these problems either do not fully addressthese drawbacks or create their own additional drawbacks. Oneoften-cited approach is to employ a sustained release delivery system ofa pharmacologically active anti-cancer agent. It is believed thatlocally administered, sustained release delivery systems allow highdoses of the anti-cancer agent to be delivered while ensuring sufficientretention at the target site. Theoretically, such an approach would bothincrease efficacy and limit toxicity. To date, however, such approacheshave not been effective. Thus additional agents and formulations areneeded in order to bring the full potential of local chemotherapy tofruition.

Recently, there has been a significant interest in Golgi apparatusdisturbing agents, particularly brefeldin A, due to its reportedanti-tumor activity. Brefeldin A (BFA) was first described to be anantifungal, cytotoxic, and cancerostatic antibiotic. Haerri, et al.(1963) Chem. Abs.59:5726h. Brefeldin A was also reported to haveanti-viral properties. Tamura et al. (1968) J. Antibiotics 21:161-166.In recent years, brefeldin A has been studied extensively as a proteintransport inhibitor. It is believed that brefeldin A can reversiblydisrupt the Golgi apparatus, thereby affecting protein transport throughthe cytoplasm. Domes et al. (1989) J. Cell Biol. 109:61-72 (1989);Lippincott-Schwartz et al. (1991) J. Cell Biol. 112:567-577. It is nowknown that brefeldin A induces retrograde membrane transport from Golgito the endoplasmic reticulum (ER). Dinter et al.(1998) Histochem. CellBiol. 109:571-590. Currently brefeldin A is primarily used as a tool byresearchers to interfere with the processing and sorting of finishedproteins in order to more fully understand protein trafficking.

Due to solubility and related toxicity problems of brefeldin A, it hasnot yet been used successfully as an active agent in a pharmaceuticalformulation. U.S. Pat. No. 4,608,078 to Acker et al. reportedpreparation of derivatives of brefeldin A in order to overcomesolubility problems, but these derivatives still exhibited toxicity andinsufficient solubility. In 1997, preparation and antitumor activity ofwater-soluble derivatives of brefeldin A were disclosed in U.S. Pat. No.5,696,154 to Malspeis et al. These derivatives were claimed to besuitable for intravenous delivery in animals and humans. However, theseanalogs have been tested only in vitro and in very small amounts, whichmay not produce desired therapeutic effects in vivo. Thus, there remainsa need for pharmaceutical formulations which can deliver, inter alia,insoluble or slightly soluble active agents such as brefeldin A andother Golgi apparatus disturbing agents, for the treatment of cellularproliferative diseases. There is a further need for pharmaceuticalformulations of brefeldin A.

SUMMARY OF THE INVENTION

Accordingly, it is a primary object of the invention to address theaforementioned needs in the art and provide a pharmaceutical formulationcomprising a Golgi apparatus disturbing agent as will be described indetail herein.

It is another object of the invention to provide a pharmaceuticalformulation comprising brefeldin A as will be described in detailherein.

It is yet another object of the invention to provide such a formulationwhich significantly reduces the drawbacks associated with other types oflocal chemotherapy.

It is still another object of the invention to provide such aformulation which provides for sustained release of the active agent.

It is yet another object of the invention to provide such a formulationwhich increases the solubility of an otherwise insoluble or slightlysoluble active agent.

It is a further object of the invention to provide a method of treatinga cellular proliferative disease comprising administering to a patientin need thereof the pharmaceutical formulation of the invention asdescribed in detail herein.

Additional objects, advantages and novel features of the invention willbe set forth in part in the description which follows, and in part willbecome apparent to those skilled in the art upon examination of thefollowing, or may be learned by practice of the invention.

In a first embodiment, then, a pharmaceutical formulation for treating acellular proliferative disease in a patient is provided comprising: atherapeutically effective amount of a Golgi apparatus disturbing agent;a biocompatible carrier; and a solvent.

The Golgi apparatus disturbing agent can be any agent known to interferewith the functioning of the Golgi apparatus but it is preferred that theGolgi disturbing apparatus disturbing agent is brefeldin A. Thebiocompatible carrier can comprise any substantially non-antigenic andnon-toxic compound that can serve to increase the solubility of theactive agent and/or provide a sustained release profile of the activeagent following administration of the pharmaceutical formulation.Preferred carriers include polysaccharides and particularly preferredpolysaccharides are selected from the group consisting of chitin,chitosan, and combinations thereof. It is preferred also that thebiocompatible carrier is covalently linked to brefeldin A.

In another embodiment, a method of treating a cellular proliferativedisease is provided comprising administering to a patient in needthereof a pharmaceutical formulation comprising: a therapeuticallyeffective amount of a Golgi apparatus disturbing agent; a biocompatiblecarrier; and a solvent.

The method of treating includes injecting the pharmaceutical formulationdirectly or nearly directly to the target site (e.g., an area exhibitingcellular proliferative disease). Preferably, the method is useful fortreating proliferative selected from the group consisting of braincancer, bladder cancer, breast cancer, colorectal cancer, head and neckcancer, liver cancer, prostate cancer, and ovarian cancer.

BRIEF DESCRIPTION OF THE FIGURES

The file of this patent contains at least one drawing executed in color.Copies of the patent with color drawing(s) will be provided by thePatent & Trademark Office upon request and payment of the necessary fee.

FIG. 1 is a graph depicting the change in tumor size with a brefeldin Aformulation of the present invention, as evaluated in Example 1.

FIGS. 2a and 2 b are color photographs of treated and untreated mice atday 0 and at day 2 as evaluated in Example 1.

DETAILED DESCRIPTION OF THE INVENTION

I. Overview and Definitions

Before describing the present invention in detail, it is to beunderstood that unless otherwise indicated this invention is not limitedto specific formulation components, manufacturing methods, dosageregimens, or the like, as such may vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to a “solvent” includes a combination of two or more solvents,etc.

In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions set forthbelow.

The terms “pharmacologically active agent,” “active agent,” and “drug”are used interchangeably herein to refer to a chemical material orcompound which, when administered to an organism (human or animal)induces a desired pharmacologic effect. Included are derivatives andanalogs of those compounds or classes of compounds specificallymentioned which also induce the desired pharmacologic effect. The activeagents herein are generally used in the treatment of cellularproliferative diseases.

The term “cellular proliferative disease” is intended to refer to anycondition characterized by the undesired propagation of cells. Includedare conditions such as neoplasms, cancers, and tumors. Also contemplatedas “cellular proliferative diseases” are non-cancerous conditions suchas benign melanomas and other cellular growths occurring within theepidermal layers.

The term “sustained release” as used herein refers to a drug formulationthat provides for gradual release of drug over an extended period oftime, and that preferably, although not necessarily, results insubstantially constant levels at the desired site over an extendedperiod of time.

“Vehicles” as used herein refer to materials suitable for drugadministration. Vehicles useful herein include any such materials knownin the art, e.g., any liquid, gel, solvent, liquid diluent, solubilizer,or the like, which is nontoxic and which does not interact with othercomponents of the composition in a deleterious manner.

By the terms “effective amount” or “therapeutically effective amount” ofan agent as provided herein are meant a sufficient amount of the agentto provide the desired therapeutic effect. Of course, undesirableeffects (e.g., side effects) are sometimes manifested along with thedesired therapeutic effect; hence a practitioner balances the potentialbenefits against the potential risks in determining what is anappropriate “therapeutically effective amount.” As will be pointed outbelow, the exact amount required will vary from subject to subject,depending on the species, age, and general condition of the subject,mode of administration, and the like. Thus, it is not possible tospecify an exact “effective amount. ” However, an appropriate “effectiveamount” in any individual case may be determined by one of ordinaryskill in the art using only routine experimentation.

The terms “biocompatible” and “pharmacologically acceptable” as usedherein are meant to refer to a material which is not biologically orotherwise undesirable, i.e., the material may be administered to anindividual along with the selected active agent without causing anyundesirable biological effects or interacting in a deleterious mannerwith any of the other components of the pharmaceutical composition inwhich it is contained. Similarly, a “pharmacologically acceptable” saltor a “pharmacologically acceptable” ester of a compound as providedherein is a salt or ester which is not biologically or otherwiseundesirable.

The terms “treating” and “treatment” as used herein refer to reductionin severity and/or frequency of symptoms, elimination of symptoms and/orunderlying cause, prevention of the occurrence of symptoms and/or theirunderlying cause, and improvement or remediation of damage. Thus, forexample, the present method of “treating” a cellular proliferativedisease, as the term “treating” is used herein, encompasses bothprevention of cellular proliferative diseases in a predisposedindividual and treatment of cellular proliferative diseases in aclinically symptomatic individual.

“Optional” or “optionally” means that the subsequently describedcircumstance may or may not occur, so that the description includesinstances where the circumstance occurs and instances where it does not.

“Patient” as used herein refers to a mammalian, preferably humanindividual who can benefit from the pharmaceutical formulations of thepresent invention. There is no limitation on the type of mammal whichcould benefit from the presently described pharmaceutical formulations.

II. The Pharmaceutical Formulation

In a first embodiment, the invention provides a pharmaceuticalformulation for treating a cellular proliferative disease comprising: atherapeutically effective amount of a Golgi apparatus disturbing agent;a biocompatible carrier; and a solvent.

A. Active Agent

It is particularly preferred that the active agent incorporated in thepresent invention is a Golgi apparatus disturbing agent. Golgi apparatusdisturbing agents include those agents which can totally disrupt theentire Golgi network or simply block one of the protein traffickingpathways. Many Golgi apparatus disturbing agents are reversible, thatis, once the agent is removed the Golgi apparatus functions normally.Although it is not entirely clear how Golgi apparatus disturbing agentsinterfere with the processing and sorting of finished proteins, more isknown about the Golgi apparatus itself. The Golgi apparatus is found inall eukaryotic cells and is responsible for receiving the proteins fromthe endoplasmic reticulum. Once arriving at the Golgi apparatus theproteins can be modified and/or sorted for delivery to the requiredlocation within the cell. For example, the Golgi apparatus isresponsible for adding sulfates to the amino acid tyrosine in certainproteins and cleaving various protein precursors to yield maturehormones and neurotransmitters to name but a few of its functions. Giventhe importance of the Golgi apparatus, it is clear how Golgi apparatusdisturbing agents can serve as powerful and important therapeuticagents.

Although any Golgi apparatus disturbing agent can be included in thepresent invention, preferred Golgi apparatus disturbing agents includethose selected from the group consisting of brefeldin A, nocodazole,ilimaquinone, bafilamycin, okadaic acid, retinoic acid and combinationsthereof. Most preferred of the Golgi apparatus disturbing agents isbrefeldin A.

Golgi apparatus disturbing agents (or any other active agent) may bepresent in the formulation as a salt, ester, amide, or other derivative,or may be functionalized in various ways as will be appreciated by thoseskilled in the art and as described in the pertinent texts, patents, andliterature; however it is preferred that the formulation containbrefeldin A per se, i.e., not in its derivatived or functionalized form.The active agents of the present invention can either be synthesizedusing techniques well known in the art or obtained from commercialsuppliers.

Golgi apparatus disturbing agents (or any other active agentincorporated into presently described pharmaceutical formulation) mayexist in the formulation in the form of a salt, ester, amide, prodrug,derivative, or the like, provided the salt, ester, amide, prodrug orderivative is suitable pharmacologically, i.e., effective in treatingits intended cellular proliferative disease. Salts, esters, amides,prodrugs and other derivatives of the active agents may be preparedusing standard procedures known to those skilled in the art of syntheticorganic chemistry and described, for example, by J. March, AdvancedOrganic Chemistry: Reactions, Mechanisms and Structure, 4th Ed. (NewYork: Wiley-Interscience, 1992). For example, acid addition salts areprepared from the free base using conventional methodology, and involvesreaction with a suitable acid. Generally, the base form of the drug isdissolved in a polar organic solvent such as methanol or ethanol and theacid is added thereto. The resulting salt either precipitates or may bebrought out of solution by addition of a less polar solvent. Suitableacids for preparing acid addition salts include both organic acids,e.g., acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalicacid, malic acid, malonic acid, succinic acid, maleic acid, fumaricacid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelicacid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,salicylic acid, and the like, as well as inorganic acids, e.g.,hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like. An acid addition salt may be reconvertedto the free base by treatment with a suitable base. Particularlypreferred acid addition salts of the active agents herein are halidesalts, such as may be prepared using hydrochloric or hydrobromic acids.Conversely, preparation of basic salts of acid moieties which may bepresent on a active agent molecule are prepared in a similar mannerusing a pharmaceutically acceptable base such as sodium hydroxide,potassium hydroxide, ammonium hydroxide, calcium hydroxide,trimethylamine, or the like. Particularly preferred basic salts hereinare alkali metal salts, e.g., sodium salt, and copper salts. Preparationof esters involves functionalization of hydroxyl and/or carboxyl groupswhich may be present within the molecular structure of the drug. Theesters are typically acyl-substituted derivatives of free alcoholgroups, i.e., moieties which are derived from carboxylic acids of theformula RCOOH where R is alkyl, and preferably is lower alkyl. Esterscan be reconverted to the free acids, if desired, by using conventionalhydrogenolysis or hydrolysis procedures. Amides and prodrugs may also beprepared using techniques known to those skilled in the art or describedin the pertinent literature. For example, amides may be prepared fromesters, using suitable amine reactants, or they may be prepared from ananhydride or an acid chloride by reaction with ammonia or a lower alkylamine. Prodrugs are typically prepared by covalent attachment of amoiety which results in a compound that is therapeutically inactiveuntil modified by an individual's metabolic system.

The amount of active agent in the formulation is preferably a unitdosage and is present in an amount of from about 0.01 mg to about 10 g.It is particularly preferred that the active agent is present in anamount of from about 0.1 mg to about 1000 mg. Most preferably, theactive agent is present in an amount of from about 1 mg to about 100 mg.

When the active agent in the formulation is brefeldin A, the formulationis preferably a unit dosage formulation. In such a formulation,brefeldin A is preferably present in an amount of from about 0.01 mg toabout 10 g. It is particularly preferred that brefeldin A is present inan amount of from about 0.1 mg to about 1000 mg. Most preferably,brefeldin A is present in an amount of from about 1 mg to about 100 mg.

B. Biocompatible Carrier

The biocompatible carrier can comprise any substantially non-antigeniccompound that can serve to increase the solubility of the active agentand/or provide a sustained release profile of the active agent followingadministration of the pharmaceutical formulation. Preferred carriers areselected from the group consisting of polysaccharides, salts ofpolysaccharides, microspheres of polysaccharides, dextrins, gums,celluloses, silicones, liposomes and combinations thereof. It isparticularly preferred that the biocompatible carrier is apolysaccharide. It is particularly preferred that the polysaccharide isselected from the group consisting of chitin(poly-N-acetyl-D-glucosamine), chitosan (deacetylated chitin), andcombinations thereof. Of course derivatives of these carriers arecontemplated as well, including, for example, derivatives formed byadding or removing sugar molecules (i.e. by increasing or decreasing theweight average molecular weight of the carrier). Thus preferred carriersmay have a weight average molecular weight of from about 500 daltons toabout 100,000 daltons, more preferably between about 10,000 daltons and60,000 daltons.

Optimally, the biocompatible carrier can be covalently bonded to theactive agent producing a carrier-active agent conjugate. For example,chitin can be covalently bonded to brefeldin A, typically through the1-OH or 13-OH moieties of the active agent. Other types of bonds (e.g.,ionic, van der Waals, etc.) between the carrier and active agent arecontemplated as well. Thus, in some embodiments of the presentinvention, it is preferred that the biocompatible polymer is covalentlylinked to brefeldin A. Methods of producing these conjugates are wellknown to those skilled in the art, and described in detail in thepertinent texts and literature.

The amount of the biocompatible carrier present in the formulation willvary depending on the particular active agent and carrier used. Ofcourse, the amount of biocompatible carrier present will also depend onother factors, such as whether the formulation is intended to contain asingle dose or multiple doses and the injectability (e.g., viscosity) ofthe resulting formulation. In addition the amount of the carrier shouldbe considered in light of the target area's environment in which thepharmaceutical formulation will be administered. For example, it isdesirable that low viscosity organs such as the prostate, brain orbladder receive a low viscosity formulation, while dense tissueexemplified by solid tumors and breast tumors should receive a highviscosity pharmaceutical formulation. These and other factors areroutinely considered by those skilled in the art. Thus, the amount ofbiocompatible carrier to be included in the pharmaceutical formulationcan be established by those skilled in the art without undueexperimentation.

The amount of biocompatible carrier contained in the formulation ispreferably present in an amount of from about 0.01% w/v to about 50% w/vof the total formulation. It is particularly preferred that thebiocompatible carrier is present in an amount of from about 0.5% w/v toabout 30% w/v. Most preferably, the biocompatible carrier is present inan amount of from about 1% w/v to about 20% w/v. When the carrier ischitin, chitosan, or a combination thereof, it is preferred that thecarrier be present in an amount of from about 1% to about 20% w/v of theentire formulation.

C. Solvent

The Golgi apparatus disturbing agent and biocompatible carrier arepresent in a pharmaceutically acceptable solvent. The solvent may beaqueous. Aqueous solvents include, for example, water, saline,liposomes, oil-in-water emulsions and the like. Alternatively, thesolvent may be non-aqueous. Non-aqueous solvents include, for example,1-butanol, 2-butanol, ethanol, ethyl ether, ethyl formate, ethylacetate, methyl acetate, 3-methyl-1 -butanol, isobutyl acetate,isopropyl acetate, 2-methyl-1-propanol, methylethyl ketone,dimethylacetamide (DMA), 1,1-dimethyloxymethane, 2,2-dimethyloxypropane,propylene glycol and water-in-oil emulsions and the like. The solventmay be an organic solvent or an inorganic solvent. As will be readilyappreciated by one skilled in the art, the choice of solvent will dependon the desired solubility, the nature of the agents comprising theformulation, and the desired release characteristics.

The amount of solvent present in the formulation will vary depending onthe type of tumor (solid or soft), solubility of active agent, thesolvent chosen, and desired form of the formulation (i.e. solution,suspension, etc.) involved. Of course, the amount of solvent presentwill also depend on other factors, such as whether the formulation isintended to contain a single dose or multiple doses. Such factors areroutinely considered by those skilled in the art. Thus, the amount ofsolvent to be included in the pharmaceutical formulation can beestablished by those skilled in the art without undue experimentation.

The amount of solvent contained in the formulation is preferably presentin an amount of from about 0.01% by volume to about 50% by volume (v/v)of formulation. It is particularly preferred that the solvent is presentin an amount of from about 0.1% by volume to about 30% by volume offormulation. Most preferably, the solvent is present in an amount offrom about 1% by volume to about 10% by volume of formulation. When thesolvent used in the formulation is chosen from the group consisting ofdimethylacetamide, 1,1-dimethyloxymethane, 2,2-dimethyloxypropane,propylene glycol, and combinations thereof, it is preferred that thesolvent is present in an amount of from about 0.1% by volume to about 5%by volume of the formulation.

D. Delivery Forms and Additional Components

The pharmaceutical formulations of the present invention can be anyart-known form. A non-limiting list of preferred delivery forms of thepresent invention include solution, suspension, dispersion, emulsion,microspheres and similar formulations.

As will be readily appreciated by those skilled in the art, the Golgiapparatus disturbing agent incorporated in the present invention can besubstituted for or added with any other class of active agent whichinterferes with cellular proliferation. Classes of active agentssuitable for inclusion in the present invention (either alone or as anadditional agent) include but are not limited to agents selected fromthe group consisting of agents causing apoptosis (cell death), agentspreventing RNA synthesis, agents that interfere with protein transport,alkylating agents, anesthetic agents, antiangiogenic agents,antibacterial agents, anti-bodies, anti-fungal agents, anti-metaboliteagents, anti-neoplastic agents with known anti-proliferative activity,anti-tumor antibiotics, anti-viral agents, biologically active peptides,biologically active proteins, chemotherapeutic agents, chimeric peptidesand proteins, cytokines, endostatin agents, fusion proteins, Golgiapparatus disturbing agents, interferons, monoclonal antibody-toxins,oligonucleotides, pain-relieving agents, plant alkaloids, signaltransduction inhibitors, and signal transduction pathway inhibitors suchas inhibitors of kinases and phosphatases and combinations thereof

Specific examples of active agents which are suitable for inclusion inthe present invention (either alone or as an additional agent) includebut are not limited to agents selected from the group consisting ofconsisting of actinomycin-D, aldesleukin, aminoglutethimide, amsacrine,anastozole, L-asparaginase, 5-azacytidine, aziridinylbenzoquinone (AZQ),bafilamycin, bioallenthirn, bleomycin, bicalutamide, brefeldin A,buserelin, busulfan, carboplatin, carmustine (BCNU), chlorambucil,cisplatin (cis-DDP), cladribine, colchicinefosfamide, cyclophosphamide,cyproterone, cytarabine (Ara-C), dacarbazine, dactinomycin,daunorubicin, deoxycoformycin, diethylstilbestrol, docetaxel,doxorubicin, doxycycline, epirubicin, estramustine, etoposide (VP-16),fludarabine, fludrocortisone, 5-fluorodeoxyuridine, 5-fluorouracil(5-FU), fluoxymesterone, flutamide, gemcitabine, genistein, goserelin,hydroxyurea, idarubicin, ifosfamide, ilimaquinone, α-interferon,irinotecan, leucovorin, leuprolide, levamisole, lomustine (CCNU),mechlorethamine, medroxyprogesterone, megestrol, melphalan,mercaptopurine (6-MP), mesna, methotrexate (MTX), minocycline,mithramycin, mitomycine, mitotane, mitoxantrone, nilutamide, nocodazole,okadaic acid, octreotide, paclitaxel, pentostatin, plicamycin, porfimer,procarbazine, retinoic acid, staurosporine, streptozocin, suramin,tamoxifen, tautomycin, teniposide (ETP), testolactone, 6-thioguanine(6-TG), thiotepa, topotecan, tyrphostins, vinblastine (VLB), vincristine(VCR), vindesine, vinorelbine, wortmannin, derivatives thereof andcombinations thereof.

The pharmaceutical formulation may include one or more additionalcomponents. Additional components include, for example, anti-microbials,buffers, antioxidants, and tonicity agents. Anti-microbial agents areused to deter the growth of microorganisms, particularly in multipledose formulations (i.e. formulation for a multiple dose vial). Suitableanti-microbial agents include, phenylmercuric nitrate, thimerosal,benzethonium chloride, benzalkonium chloride, phenol, cresol,chlorobutanol, and combinations thereof. Buffers are used primarily tostabilize a solution against potential chemical degradation resultingfrom a change in the formulation's pH. Suitable buffers include acidsalts of citrates, acetates, and phosphates. Antioxidants are used topreserve products because of the ease with which drugs may oxidize.Examples of antioxidants include sodium bisulfite, acetone sodiumbisulfite, sodium formaldehyde, sulfoxylate, and thiourea, sodium saltof ethylenediaminetetraacetic acid, and substitution with an inert gas(e.g, nitrogen). Tonicity agents are used to control tonicity to enurethat the resulting formulation is not excessively hypotonic orhypertonic relative to the physiological state. Examples of tonicityagents include electrolytes (e.g. sodium chloride), and mono- ordisaccharides (e.g., dextrose for monosaccharides).

The amount of additional components which are not active agents willvary depending on the solvents chosen, desired form of the formulationand other factors. Such factors are routinely considered by thoseskilled in the art. Thus the amount of additional components which arenot active agents can be established by those skilled in the art withoutundue experimentation.

The amount of such components preferably will not exceed 10% by volumeof the total formulation. More preferably such components will notexceed 1% by volume of the total formulation. Most preferably suchcomponents will be present in an amount of from about 0.001% by volumeto about 1.0%.

E. Pharmacokinetic Profile

The pharmaceutical formulations of the present invention are preferablysustained release formulations. That is, the formulation releases theactive agent over an extended period of time relative to an immediaterelease or non-sustained release formulation. It is preferred that thepharmaceutical formulations of the present invention release activeagent over a time of from about 4 hours to about 24 hours.

III. Methods of Treatment

The present invention also provides a method of treating a cellularproliferative disease comprising administering to a patient in needthereof a pharmaceutical formulation comprising: a therapeuticallyeffective amount of a Golgi apparatus disturbing agent; a biocompatiblecarrier; and a solvent.

It is preferred that the method of administering the pharmaceuticalformulation to the patient is via local injection. Those skilled in theart will recognize that delivery via injection contemplates the use of asyringe, catheter or similar device, which delivers the pharmaceuticalformulation of the invention to the target site, i.e., to an areaexhibiting cellular proliferative disease. Delivery may be direct, i.e.,intratumoral, or nearly direct, i.e., intralesional, that is, to an areathat is sufficiently close to a tumor so that the active agent exhibitsthe desired pharmacological activity with respect to the tumor itself.Thus, the pharmaceutical formulations are preferably deliveredintralesionally or intratumorally. In addition, it is preferred that thepatient is human. It is also preferred that the patient is a mammal.

The amount of the active agent administered will, of course, bedependent on the subject being treated, the subject's weight, the mannerof administration and the judgement of the prescribing physician. In themethod of the invention, i.e., preventing or treating a cellularproliferative disease, an initial dosing schedule will generally involveadministering doses of from about 0.01 mg/kg to about 10 g/kg of thepatient's body weight. It is particularly preferred that the activeagent is administered in an amount of from about 0.1 mg/kg to about 1000mg/kg of the patient's body weight. Most preferably, the active agent isadministered in an amount of from about 1 mg/kg to about 100 mg/kg ofthe patient's body weight. Depending on the response of the disease,additional dosages within this range can be administered in order totreat the disease.

The total amount of the formulation delivered to the diseased site willdepend on, inter alia, the size, extent and type of tumor and thepatient's body weight. Generally, it is preferred that the total volumeadministered is between about 0.1 mL to about 60 mL and more preferablybetween about 0.5 mL to about 30 mL. Most preferably, the total volumeadministered of the presently described pharmaceutical formulation isfrom about 1.0 mL to about 15 mL.

It is preferred that the pharmaceutical formulations of the presentinvention be directed to the target area with the assistance of a CT,ultrasound, or similar device in order to ensure correct placement. Oncethe initial dose is administered, the patient may be given other doseseither immediately or after a period of time. Such a dosing schedule caneasily be determined by one skilled in the art after taking intoconsideration the nature of the disease, strength of the patient,expected effects of the formulation, etc.

It is intended that by local delivery of the presently describedpharmaceutical formulation, higher concentration of the active agent canbe retained at the target site. There are several advantages to havinghigh concentrations delivered directly at the target site. First, sincethe active agent is localized, there is less potential for toxicity tothe patient since little systemic circulation occurs. Second, drugefficacy is improved since the target site is exposed to higherconcentrations of drug. Third, the relatively fast delivery ensures bothsolubility of the drug and little or no degradation of the active agentbefore reaching the target site. Fourth, the method is non-invasive,which is ideal for unresectable tumors such as brain tumors, livertumors, and pancreatic tumors.

The cellular proliferative disease that can be treated using the presentmethods and formulations will, of course, depend on the active agentwhich is incorporated into the pharmaceutical formulation. Generallyspeaking, cellular proliferative diseases comprise all sarcomas,carcinomas, lymphomas, and malignant melanomas but also include, forexample, non-cancerous melanomas and other benign growths caused byrapidly dividing cells.

Non-limiting examples of cellular proliferative diseases for which thepresent invention is suited include adrenocorticol cancer, bladdercancer, bone cancer, brain cancer, breast cancer, cervical cancer, coloncancer, colorectal cancer, endometrial cancer, esophogeal cancer, eyecancer, gallbladder cancer, gastric cancer, head and neck cancer,laryngeal cancer, liver cancer, lung cancer, melanoma,myeloproliferative disorders, neck cancer, nonmelanoma skin cancer,ovarian cancer, prostate cancer, pancreatic cancer, rectal cancer, andtesticular cancer. Diseases for which the present invention isparticularly well suited to treat and are therefore preferred includethose diseases selected from the group consisting of brain cancer,bladder cancer, breast cancer, colon cancer, colorectal cancer, rectalcancer, head and neck cancer, liver cancer, prostate cancer and ovariancancer.

It is to be understood that while the invention has been described inconjunction with the preferred specific embodiments thereof, that thedescription above as well as the examples which follow are intended toillustrate and not limit the scope of the invention. Other aspects,advantages and modifications within the scope of the invention willbecome apparent to those skilled in the art to which the inventionpertains.

All patents, patent applications, and publications mentioned herein,both supra and infra are hereby incorporated herein by reference.

EXPERIMENTAL

For each of the following examples, the pharmaceutical formulations wereprepared just prior to use. Various amounts of brefeldin A (mg ofdrug/kg animal's body weight) were weighed out and varying amounts ofsolvent was used to mix the drug. The mixtures were either vortexed(mechanical mixing) or sonicated until drug was either completelysoluble or only a very small amount appeared to settle in the bottom thetest tube. Chitin was then added and the mixture was sonicated.

In vivo activity was determined using nude mice carrying a human tumor.The human oral epithelial (KB-1) cells were kept in CO₂/O₂ atmosphere at37° C., the cells were grown in monolayers, when confluent, cells wererinsed with ice cold phosphate buffered/EDTA, followed by Trypsin/EDTAtreatment for about 3 minutes at 37° C. The cells were then dispersed bythe addition of media with serum and spun down using a centrifuge andwashed once in Phosphate Buffered Saline (PBS), prior to suspending atthe desired density (1×10⁷ cells/mL) in sterile Hanks Balance SaltSolution (HBSS). The cells were then inoculated with a 100 ul injectionon to each flank of the mice to provide 1×10⁶ cells per site.

Six days following inoculation, the mice normally grew a tumor on eachflank, ranging in size from 100 to 120 mm³. The mice (average weight=25g) were randomized and separated into groups. Each tumor was thenmeasured and the tumor volume calculated and recorded. In experimentalgroups, one tumor on each mouse was treated with the appropriate agent(drug versus drug in formulation) with an injection volume of 50 ulusing a 22G½ needle. The control group was left untreated.

Antitumor activity was determined by inhibition of tumor growth andsurvival rate. Each tumor was measured two to three times per week. Innude mice, tumors were measured for 10 days or until mice survived. InBalb/C tumors were measured for up to 20 days following treatment. Thevolume of the tumor was calculated and recorded. Tumor volume data isanalyzed by using simple regression model.

Example 1 In vivo Efficacy of BFA, Chitin and Dimethylacetamide (DMA) inNude Mice Carrying Human Epithelial (KB-1) Tumors

Nude mice were divided into groups and given treatments according toTable 1.

TABLE 1 # of treat- # of Lethal Grp. Treatment ments Dose tumorstoxicity 1 Untreated —  ,  ,   6 0 2 DMA/BFA 1 25 μl, 0.5 mg,   4 0 3DMA/BFA/Chitin 1 25 μl, 0.5 mg, 1 mg 4 0

Results

A higher degree of efficacy was demonstrated when brefeldin A wasdelivered with dimethylacetamide (DMA) and chitin(poly-N-acetyl-D-glucosamine), as compared to brefeldin A (BFA) withDMA. This three component formulation was extremely effective in tumorcontrol: an acute response of blackening and almost total elimination ofthe tumor with one treatment of both BFA/DMA and BFA/DMA/chitin wasobserved. However, this response was more prominent in the sustainedformulation (i.e., BFA/DMA/Chitin).

The following results were obtained. Vol represents the average tumorvolume in mm³ in a given group. % represents the normalized tumor volumefrom day 0.

Day Day Day Day Day Day Day Day 0 0 2 2 6 6 9 9 Group Vol. % Vol. % Vol.% Vol. % Control 83.7 100.0 182.3 217.8 405.9 484.9 481.0 574.7 DMA +BFA 49.1 100.0 96.9 197.4 107.5 218.9 125.1 254.8 DMA + 152.0 100.0 86.256.7 147.9 97.3 159.1 104.7 BFA + Chitin

A graph as seen in FIG. 1 depicts the change in tumor size with acontrol and a sustained release formulation of brefeldin A. Not onlywere the brefeldin A formulations useful in treating the tumor, theywere effective as well.

As seen in the top photographs in FIGS. 2a and 2 b all mice at day 0exhibited intradermal human epithelial (KB-1) tumors.

As seen in the bottom photograph of FIG. 2b mice which were treated withbrefeldin A, chitin and DMA showed dramatic improvement within 48 hoursin their lesions than the mice which were untreated (bottom photographof FIG. 2b). Thus, in vivo efficacy was established.

Example 2 In vivo Efficacy of Brefeldin A, Chitin and Varying Amount ofDimethylacetamide (DMA) in Balb/C Mice Carrying Erlich Tumors

Balb/C mice are divided into groups and given treatments according toTable 2.

TABLE 2 # of treat- # of Lethal Grp. Treatment ments Dose tumor toxicityT/C 1 Untreated —  ,  ,   6 0 — 2 DMA 1 20 μl,  ,   6 0 1.20 3 DMA 1 10μl,  ,   6 0 1.15 4 DMA 1 5 μl,  ,   6 0 1.10 5 DMA/BFA 1 20 μl, 0.5 mg,  6 0 1.75 6 DMA/BFA 1 10 μl, 0.5 mg,   6 0 1.40 7 DMA/BFA 1 5 μl, 0.5mg,   6 0 1.25 8 DMA/BFA/ 1 20 μl, 0.5 mg, 1 mg 6 0 2.75 Chitin 9DMA/BFA/ 1 10 μl, 0.5 mg, 1 mg 6 0 2.50 Chitin 10 DMA/BFA/ 1 5 μl, 0.5mg, 1 mg 6 0 2.00 Chitin T/C: ratio of delay in tumor growth in responseto treatment (T) vs. untreated control (C).

Results

A high T/C ratio (2.00-2.75) of delay in tumor growth for groups 8, 9,and 10 demonstrates a significant increase in the efficacy of brefeldinA when delivered with DMA and chitin.

In groups 5, 6, and 7, where brefeldin A is given with the solvent, alower T/C ratio (1.25-1.75) is obtained and even lower T/C ratios(1.10-1.20) are obtained in groups 2, 3, and 4, where mice are treatedwith different amounts of DMA.

Example 3 Survival of Balb/C Mice Following Treatment with Brefeldin A,DMA, and Chitin, Separately and in Combination

Balb/C mice are divided into groups and given treatments according toTable 3. Mice are observed daily for 25 days for deaths due to toxicity.

TABLE 3 # of treat- # of # of Grp. Treatment ments Dose tumors Deaths 1Untreated —  ,  ,   10 0 2 DMA/ /  1 30 μl,  ,   10 2 3 DMA/ /Chitin 130 μl,  , 1 mg 10 0 4 DMA/BFA/  1 30 μl, 2 mg,   10 4 5 DMA/BFA/Chitin 130 μl, 2 mg, 1 mg 10 1 6 DMA/ /  1 10 μl,  ,   10 0 7 DMA/ /Chitin 1 10μl,  , 1 mg 10 0 8 DMA/BFA/  1 10 μl, 1 mg,   10 2 9 DMA/BFA/Chitin 1 10μl, 1 mg, 1 mg 10 0

Results

Administration of high doses of brefeldin (80 mg/kg and 40 mg/kg) withDMA (80% and 20%) in groups 4 and 8, are proved to be most toxic tomice. However, when chitin is added to the formulation, less toxicity tomice is observed, most likely due to the sustained release effect.

Example 4 In vivo Efficacy of Brefeldin A, DMA with Varying Amount ofChitin in Balb/C Mice Carrying Erlich Tumors

Balb/C mice are divided into groups and given treatments according toTable 4. Mice are observed daily to evaluate lethal toxicity and tumorsare measured on alternate days for 20 days.

TABLE 4 # of treat- # of Lethal Grp. Treatment ments Dose tumor toxicityT/C 1 Untreated —  ,  ,   6 0 — 2 DMA 1 10 μl,  ,   6 0 1.17 3 DMA/BFA/1 10 μl, 2 mg,   6 1 2.50   4 DMA/BFA/ 1 10 μl, 2 mg, 1 mg 6 0 2.85Chitin 5 DMA/BFA/ 1 10 μl, 2 mg, 0.5 mg 6 1 2.77 Chitin 6 DMA/BFA/ 1 10μl, 2 mg, 0.25 6 1 2.55 Chitin mg 7 DMA/BFA/ 1 10 μl, 0.5 mg, 1 mg 6 02.55 Chitin 8 DMA/BFA/ 1 10 μl, 0.5 mg, 0.5 6 0 2.40 Chitin mg 9DMA/BFA/ 1 10 μl, 0.5 mg, 0.25 6 0 2.30 Chitin mg T/C: ratio of delay intumor growth in response to treatment (T) vs. untreated control (C).

Results

Toxic effects are observed with high doses of brefeldin A whenadministered with DMA and low amounts of chitin (3, 5, and 6). Howeverthis toxicity lessens when 1.0% to 2.0% of chitin is included in theformulation, groups 4, 5, 8, and 9.

A high T/C ratio (2.50-2.85) of delay in tumor growth for groups 4, 5,and 6 demonstrates a significant increase in the efficacy of brefeldin Awhen delivered with DMA and chitin.

When the dose of brefeldin A is decreased, as in groups 7, 8, and 9, adecrease in T/C ratio (2.30-2.55) is observed.

Example 5 In vivo Efficacy of Brefeldin A with Chitin and Ethyl Acetate(Veh A.) and Tetrahydrofuran (Veh. B) in Balb/C Mice Carrying ErlichTumors

Balb/C mice are divided into groups and given treatments according toTable 5.

TABLE 5 # of # of treat- tu- Lethal Grp. Treatment ments Dose morstoxicity T/C 1 Untreated —   6 0 — 2 Vehicle A/ /  1 5 μl,  ,   6 0 1.103 Veh. A/BFA/  1 5 μl, 0.5 mg, 6 0 1.35   4 Veh. A/BFA/ 1 5 μl, 0.5 mg,6 0 1.58 Chitin 1 mg 5 Vehicle B/ /  1 5 μl,  ,   6 0 1.08 6 Veh.B/BFA/  1 5 μl, 0.5 mg, 6 0 1.40   7 Veh. B/BFA/ 1 5 μl, 0.5 mg, 6 01.88 Chitin 1 mg T/C: ratio of delay in tumor growth in response totreatment (T) vs. untreated control (C).

Results

Mice treated with the triple component formulation (i.e. groups 4 and 7)demonstrate significantly reduced tumor size than all other groups. Nostatistical difference is demonstrated between the solvents chosen.

Example 6 In vivo Efficacy of Brefeldin A with Chitin and Ethyl Acetate(Veh. A) in Single vs. Multiple Doses in Balb/C Mice Carrying ErlichTumors

Balb/C mice are divided into groups and given treatments according toTable 6. Multiple dose groups receive injection every day for 5 days.

TABLE 6 # of # of treat- tu- Lethal Grp. Treatment ments Dose morstoxicity T/C 1 Untreated —  ,  ,   6 0 — 2 Vehicle A/ /  1 5 μl,  ,   60 1.08 3 Veh. A/BFA/  1 5 μl, 0.5 mg, 6 0 1.37   4 Veh. A/BFA/ 1 5 μl,0.5 mg, 6 0 1.54 Chitin 1 mg 5 Vehicle A/ /  5 5 μl,  ,   6 1 1.20 6Veh. A/BFA/  5 5 μl, 0.5 mg, 6 2 1.45   7 Veh. A/BFA/ 5 5 μl, 0.5 mg, 61 1.98 Chitin 1 mg T/C: ratio of delay in tumor growth in response totreatment (T) vs. untreated control (C).

Results

Toxicity is observed in groups 5, 6, and 7 with multiple doses. Micetreated with the triple component formulation (i.e. groups 4 and 7)demonstrate increased efficacy than all other groups within the samenumber of treatments. As between groups 4 and 7, the mice receiving(group 7) exhibit a statistically greater reduction in tumor volume thanmice receiving a single treatment.

Example 7 Survival of Balb/C Mice Following a Single Treatment withEthyl Acetate (Veh A), BFA/Ethyl Acetate and BFA/Ethyl Acetate/ChitinSingle vs. Multiple Dose.

Balb/C mice are divided into groups and given treatments according toTable 7.

TABLE 7 # of treat- # of # of Grp. Treatment ments Dose tumors deaths 1Untreated —  ,  ,   10 0 2 Vehicle A/ /  1 5 μl,  ,   10 0 3 Veh.A/BFA/  1 5 μl, 0.25 mg,   10 0 4 Veh. A/BFA/ 1 5 μl, 0.25 mg, 1 mg 10 0Chitin 5 Vehicle A/ /  5 5 μl,  ,   10 1 6 Veh. A/BFA/  5 5 μl, 0.05 mg,  10 1 7 Veh. A/BFA/ 5 5 μl, 0.05 mg, 1 mg 10 0 Chitin

Results

Multiple doses of ethyl acetate in groups 5 and 6 result in deaths ineach group. All other animals tolerate the treatment.

What is claimed is:
 1. A pharmaceutical formulation for treating acellular proliferative disease consisting essentially of: atherapeutically effective amount of a Golgi apparatus disturbing agentselected from the group consisting of brefeldin A, nocodazole,ilimaquinone, bafilamycin, okadaic acid and combinations thereof; abiocompatible carrier selected from the group consisting of chitin,chitosan, and combinations thereof; and a solvent.
 2. The pharmaceuticalformulation of claim 1, wherein the Golgi apparatus disturbing agent isbrefeldin A.
 3. The pharmaceutical formulation of claim 1, in unitdosage form.
 4. The pharmaceutical formulation of claim 3, wherein theGolgi apparatus disturbing agent is present in an amount of from about0.01 mg to about 10 g.
 5. The pharmaceutical formulation of claim 4,wherein the Golgi apparatus disturbing agent is brefeldin A.
 6. Thepharmaceutical formulation of claim 1, wherein the biocompatible carrieris present in an amount of from about 0.01% w/v to about 50% w/v of thetotal formulation.
 7. The pharmaceutical formulation of claim 1, whereinthe solvent is selected from the group consisting of dimethylacetamide,1,1-dimethyloxymethane, 2,2-dimethyloxypropane, propylene glycol, water,saline, 1-butanol, 2-butanol, ethanol, ethyl ether, ethyl formate, ethylacetate, methyl acetate, 3-methyl-1-butanol, isobutyl acetate, isopropylacetate, 2-methyl-1-propanol, and methyl ethyl ketone.
 8. Thepharmaceutical formulation of claim 1, wherein the solvent is present inan amount of from about 0.01% by volume to about 50% by volume (v/v) offormulation.
 9. The pharmaceutical formulation of claim 2, wherein thebiocompatible carrier is covalently linked to the brefeldin A.
 10. Thepharmaceutical formulation of claim 1, wherein the formulation providesa sustained release profile in vivo.
 11. The pharmaceutical formulationof claim 1, housed in an injection device.
 12. The pharmaceuticalformulation of claim 11, wherein the injection device comprises asyringe.
 13. The pharmaceutical formulation of claim 11, wherein theinjection device comprises a catheter.
 14. The pharmaceuticalformulation of claim 1, wherein the solvent is selected from the groupconsisting of ethers, lower alcohols, esters, ketones, alkoxyalkanes,and combinations thereof.
 15. The pharmaceutical formulation of claim 7,wherein the solvent is ethanol.